The long-term objective of the PI is to gain in-depth understanding of a specific area in cell biology such that the PI will be able to define and explore unique biomedical research opportunities using advanced optical biophysical methods. The objective of the proposed project is to understand the dynamics of the morphogenesis of breast epithelial structures by two-photon excitation and fluorescence lifetime imaging microscopy and define the processes that occur prior to and during apoptotic events associated with lumen formation. In addition, I aim to elucidate signaling pathways associated with distinct steps in the morphogenesis of hollow, spherical growth arrested mammary epithelial structures. The majority of benign and malignant breast cancers are characterized by filling of the hollow lumen of the end spheroid structure (or acinus). Preliminary evidence indicates that luminal clearance is at least partially due to selective apoptosis of inner cells due to lack of activation of critical cell signaling proteins. We have found that a dichotomy develops between the outer and inner acinar cells during morphogenesis and that the inner cells become nonresponsive to EGFR stimulation. We hypothesize that apoptosis takes place in the inner cells as a result of deprivation of signals that are important to cell survival. I will investigate the normal processes that are associated with the development of this dichotomy in order to elucidate the mechanisms responsible for the differential apoptosis of inner acinar cells and mechanism whereby tumor cells escape apoptosis and fill the luminal space. Aim 1: To define the precise spatial and temporal dynamics of events involved in acinar morphogenesis and lumen formation in order to understand the temporal and spatial relationships between these processes. Aim 2: To define the specific steps in signaling from EGFR that are defective within the inner acinar cells in order to understand the basis for the absence of signaling responses in these cells. Aim 3: To examine the dynamics of processes associated with activation of oncogenes in acinar structures by inducible activation. The proposed project addresses important issues fundamental to our understanding of normal mammary morphogenesis and breast cancer. The implementation of biophysical imaging approaches in this proposal is aimed at complementing biochemical and molecular investigations. The results from this study will provide new insights into the early events of breast cancer, lead to a better understanding how tumor cells escape cell death in the luminal space, and may provide a basis for therapeutic intervention in cancer.